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Cytotoxic diterpenoids from Salvia glutinosa and comparison with the tanshinone profile of danshen (Salvia miltiorrhiza).

The roots of Salvia miltiorrhiza are the source of the traditional Chinese medicine danshen and the class of tanshinones, particular quinoid nor-diterpenoids of the abietane type. Of these compounds, cryptotanshinone, dihydrotanshinone I, tanshinone I, and tanshinone IIA, have been extensively studied for their anticancer potential, not only but as well because of their high abundance in S. miltiorrhiza and their thus easy availability. However, also additional Salvia species are known to contain tanshinones, mainly such of the subgenus Glutinaria, of which S. glutinosa is the only species widely occurring in Europe. Using UHPLC-DAD-MS, the tanshinone profile of S. glutinosa roots collected from two different locations was compared to the profile in S. miltiorrhiza roots. In addition, tanshinone IIA and another six diterpenoids from S. glutinosa were investigated for their antiproliferative and cytotoxic potential against MDA-MB-231 and HL-60 cells. Apart from dihydrotanshinone I, which has been previously characterized due to its anticancer properties, we determined danshenol A as a highly antiproliferative and cytotoxic agent, significantly surpassing the effects of dihydrotanshinone I. With regard to the diterpenoid profile, S. miltiorrhiza showed a higher concentration for most of the tanshinones, except for (+)-danshexinkun A, which was present in comparable amounts in both species. Danshenol A, in contrast, was only present in S. glutinosa as were dehydroabietic acid and (+)-pisiferic acid. The results of our study underlines the long traditional use of danshen due to its high amount on tanshinones, but also demonstrates the potential value of investigating closely related species for the discovery of new biologically active lead compounds.

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Whole stromal fibroblast signature is linked to specific chemokine and immune infiltration patterns and to improved survival in NSCLC

ABSTRACT Cancer associated fibroblasts (CAF) are known to orchestrate multiple components of the tumor microenvironment, whereas the influence of the whole stromal-fibroblast compartment is less understood. Here, an extended stromal fibroblast signature was investigated to define its impact on immune cell infiltration. The lung cancer adenocarcinoma (LUAD) data set of the cancer genome atlas (TCGA) was used to test whole stroma signatures and cancer-associated fibroblast signatures for their impact on prognosis. 3D cell cultures of the NSCLC cancer cell line A549 together with the fibroblast cell line SV80 were used in combination with infiltrating peripheral blood mononuclear cells (PBMC) for in-vitro investigations. Immune cell infiltration was assessed via flow cytometry, chemokines were analyzed by immunoassays and RNA microarrays. Results were confirmed in specimens from NSCLC patients by flow cytometry or immunohistochemistry as well as in the TCGA data set. The TCGA analyses correlated the whole stromal-fibroblast signature with an improved outcome, whereas no effect was found for the CAF signatures. In 3D microtumors, the presence of fibroblasts induced infiltration of B cells and CD69+CD4+ T cells, which was linked to an increased expression of CCL13 and CXCL16. The stroma/lymphocyte interaction was confirmed in NSCLC patients, as stroma-rich tumors displayed an elevated B cell count and survival in the local cohort and the TCGA data set. A whole stromal fibroblast signature was associated with an improved clinical outcome in lung adenocarcinoma and in vitro and in vivo experiments suggest that this signature increases B and T cell recruitment via induction of chemokines.

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Abnormal biomarkers predict complex FAS or FADD defects missed by exome sequencing

BackgroundElevated TCRαβ+CD4-CD8- double-negative T-cells (DNT) and serum biomarkers help identifying FAS mutant patients with autoimmune lymphoproliferative syndrome (ALPS). However, in some patients with clinical features and biomarkers consistent with ALPS, germline or somatic FAS mutations cannot be identified upon standard exon sequencing (ALPS-undetermined: ALPS-U). ObjectiveWe aimed to explore whether complex genetic alterations in the FAS gene escaping standard sequencing or mutations in other FAS pathway-related genes could explain these cases. MethodsGenetic analysis included whole FAS gene sequencing, copy number variation analysis and sequencing of FAS cDNA and other FAS pathway related genes. It was guided by FAS expression analysis on CD57+DNT, which can predict somatic loss-of-heterozygosity (sLOH). ResultsNine of 16 ALPS-U patients lacked FAS expression on CD57+DNT predicting heterozygous “loss of expression” FAS mutations plus acquired somatic second hits in the FAS gene, enriched in DNT. Indeed, 7/9 analyzed patients carried deep intronic mutations or large deletions in the FAS gene combined with sLOH detectable in DNT, one patient showed a FAS exon duplication. Three patients had reduced FAS expression, two of them harbored mutations in the FAS promoter, which reduced FAS expression in reporter assays. Three of the four ALPS-U patients with normal FAS expression carried heterozygous FADD mutations with sLOH. ConclusionA combination of serum biomarkers and DNT phenotyping is an accurate means to identify patients with ALPS who are missed by routine exome sequencing. Clinical implicationDetection of complex FAS pathway gene alterations by extended genetic analysis allows targeted therapy with sirolimus.

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Preadipocytes in human granulation tissue: role in wound healing and response to macrophage polarization

BackgroundChronic non-healing wounds pose a global health challenge. Under optimized conditions, skin wounds heal by the formation of scar tissue. However, deregulated cell activation leads to persistent inflammation and the formation of granulation tissue, a type of premature scar tissue without epithelialization. Regenerative cells from the wound periphery contribute to the healing process, but little is known about their cellular fate in an inflammatory, macrophage-dominated wound microenvironment.MethodsWe examined CD45−/CD31−/CD34+ preadipocytes and CD68+ macrophages in human granulation tissue from pressure ulcers (n=6) using immunofluorescence, immunohistochemistry, and flow cytometry. In vitro, we studied macrophage-preadipocyte interactions using primary human adipose-derived stem cells (ASCs) exposed to conditioned medium harvested from IFNG/LPS (M1)- or IL4/IL13 (M2)-activated macrophages. Macrophages were derived from THP1 cells or CD14+ monocytes. In addition to confocal microscopy and flow cytometry, ASCs were analyzed for metabolic (OXPHOS, glycolysis), morphological (cytoskeleton), and mitochondrial (ATP production, membrane potential) changes. Angiogenic properties of ASCs were determined by HUVEC-based angiogenesis assay. Protein and mRNA levels were assessed by immunoblotting and quantitative RT-PCR.ResultsCD45−/CD31−/CD34+ preadipocytes were observed with a prevalence of up to 1.5% of total viable cells in human granulation tissue. Immunofluorescence staining suggested a spatial proximity of these cells to CD68+ macrophages in vivo. In vitro, ASCs exposed to M1, but not to M2 macrophage secretome showed a pro-fibrotic response characterized by stress fiber formation, elevated alpha smooth muscle actin (SMA), and increased expression of integrins ITGA5 and ITGAV. Macrophage-secreted IL1B and TGFB1 mediated this response via the PI3K/AKT and p38-MAPK pathways. In addition, ASCs exposed to M1-inflammatory stress demonstrated reduced migration, switched to a glycolysis-dominated metabolism with reduced ATP production, and increased levels of inflammatory cytokines such as IL1B, IL8, and MCP1. Notably, M1 but not M2 macrophages enhanced the angiogenic potential of ASCs.ConclusionPreadipocyte fate in wound tissue is influenced by macrophage polarization. Pro-inflammatory M1 macrophages induce a pro-fibrotic response in ASCs through IL1B and TGFB1 signaling, while anti-inflammatory M2 macrophages have limited effects. These findings shed light on cellular interactions in chronic wounds and provide important information for the potential therapeutic use of ASCs in human wound healing.

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A comprehensive study of SARS-CoV-2 main protease (Mpro) inhibitor-resistant mutants selected in a VSV-based system.

Nirmatrelvir was the first protease inhibitor (PI) specifically developed against the SARS-CoV-2 main protease (3CLpro/Mpro) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron Mpro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available PIs (nirmatrelvir and ensitrelvir) with cell-based and biochemical assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation Mpro inhibitors, will help to understand SARS-CoV-2 protease-inhibitor-resistance mechanisms and show the relevance of specific mutations in the clinic, thereby informing treatment decisions.

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Step-in dosing of bosutinib in pts with chronic phase chronic myeloid leukemia (CML) after second-generation tyrosine kinase inhibitor (TKI) therapy: results of the Bosutinib Dose Optimization (BODO) Study

The approved dose of bosutinib in chronic phase CML is 400 mg QD in first-line and 500 mg QD in later-line treatment. However, given that gastrointestinal (GI) toxicity typically occurs early after treatment initiation, physicians often tend to start therapy with lower doses although this has never been tested systematically in prospective trials in the Western world. The Bosutinib Dose Optimization (BODO) Study, a multicenter phase II study, investigated the tolerability and efficacy of a step-in dosing concept of bosutinib (starting at 300 mg QD) in chronic phase CML patients in 2nd or 3rd line who were intolerant and/or refractory to previous TKI treatment. Of 57 patients included until premature closure of the study due to slow recruitment, 34 (60%) reached the targeted dose level of 500 mg QD following the 2-weekly step-in dosing regimen. While the dosing-in concept failed to reduce GI toxicity (grade II–IV, primary study endpoint) to < 40% (overall rate of 60%; 95% CI: 45–74%), bosutinib treatment (mean dosage: 403 mg/day) showed remarkable efficacy with a cumulative major molecular remission (MMR) rate of 79% (95% CI: 66 to 88%) at month 24. Of thirty patients refractory to previous therapy and not in MMR at baseline, 19 (64%) achieved an MMR during treatment. GI toxicity did not significantly impact on patient-reported outcomes (PRO) and led to treatment discontinuation in only one patient. Overall, the results of our trial support the efficacy and safety of bosutinib after failure of second-generation TKI pre-treatment. Trial registration: NCT02577926.

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Comparative Analysis of Whole Transcriptome Single-Cell Sequencing Technologies in Complex Tissues

ABSTRACTThe development of single-cell omics tools has enabled scientists to study the tumor microenvironment (TME) in unprecedented detail. However, each of the different techniques may have its unique strengths and limitations. Here we directly compared two commercially available high-throughput single-cell RNA sequencing (scRNA-seq) technologies - droplet-based 10X Chromiumvs.microwell-based BD Rhapsody - using paired samples from patients with localized prostate cancer (PCa) undergoing a radical prostatectomy.Although high technical consistency was observed in unraveling the whole transcriptome, the relative abundance of cell populations differed. Cells with low-mRNA content such as T cells were underrepresented in the droplet-based system, at least partly due to lower RNA capture rates. In contrast, microwell based scRNA-seq recovered less cells of epithelial origin. Moreover, we discovered platform-dependent variabilities in mRNA quantification and cell-type marker annotation. Overall, our study provides important information for selection of the appropriate scRNA-seq platform and for the interpretation of published results.SYNOPSISComparison of scRNA-seq protocols uncovers disparities in RNA-to-library conversionMicrowell-based scRNA-seq technology excels in capturing low-mRNA content cellsBiased transcriptomes due to gene specific RNA detection efficacies by both platformsThe study guides in informed scRNA-seq platform selection and data interpretation

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